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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0657—Cardiomyocytes; Heart cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
  • A61L27/3633—Extracellular matrix [ECM]
• • A—HUMAN NECESSITIES
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
  • A61L27/367—Muscle tissue, e.g. sphincter
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3826—Muscle cells, e.g. smooth muscle cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
  • A61L27/3891—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types as distinct cell layers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y10/00—Processes of additive manufacturing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y70/00—Materials specially adapted for additive manufacturing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B33—ADDITIVE MANUFACTURING TECHNOLOGY
  • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
  • B33Y80/00—Products made by additive manufacturing
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
  • A61F2240/001—Designing or manufacturing processes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1329—Cardiomyocytes
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1352—Mesenchymal stem cells
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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  • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
  • C12N2537/10—Cross-linking

Definitions

• the present invention relates to a three-dimensional structure for myocardial tissue regeneration and a method of manufacturing the same. More specifically, the present invention provides a first bioprinting composition comprising a tissue-engineering construct-forming solution and cardiac progenitor cells, including decellularized extracellular matrix and a crosslinking agent . Printing and crosslinking with a second bioprinting composition comprising the tissue engineering structure-forming solution, mesenchymal stem cells and vascular endothelial growth factor to alternately arrange the first bioprint layer and the second bioprint layer to form a three-dimensional structure. Forming a; And a step of thermally gelling the crosslinked three-dimensional structure to obtain a crosslinked-gelled three-dimensional structure.
• the present invention relates to a method for producing a three-dimensional structure for myocardial tissue regeneration and a three-dimensional structure for regenerating myocardial tissue.
• Three-dimensional printing converts arbitrary shape information obtained from complex shape tissue or organ medical data into G-code and builds a complex skeletal structure through a layer-by-layer process. I say that.
• Such three-dimensional printing is also referred to as 'three-dimensional bioprinting' (3D bioprinting).
• the multi-axis long-term printing system is one of the representative three-dimensional printing technologies. It is a piston type that precisely injects nanoliter units using two pneumatic syringes and a step motor that injects the material pneumatically. It consists of two syringes, and various materials can be used at the same time.
• thermoplastic biocompatible polymers such as polylactic acid (PLA), poly-glycolic acid (PGA), poly-lactic-co-glycolid acid (PLGA), polycaprolactone (PCL), and black silver mixtures thereof are mounted in pneumatic syringes.
• PLA polylactic acid
• PGA poly-glycolic acid
• PLGA poly-lactic-co-glycolid acid
• PCL polycaprolactone
• black silver mixtures thereof are mounted in pneumatic syringes.
• a hydrogel made of collagen, hyaluronic acid, gelatin, alginate, chitosan or fibrin is used to prepare a three-dimensional structure using a piston syringe. Since bioprinting requires the distribution of cell-containing media, an important aspect of bioprinting is that the printing process must be cytocompatible. This limitation reduces the choice of materials because of the need to be performed in an aqueous or aqueous gel environment.
• hydrogels using materials such as gelatin, gelatin / chitosan, gelatin / alginate, gelatin / pipronectin, lutrol F127 / alginate, and alginate can be used for various tissues from liver to bone. It is used in bioprinting for manufacturing.
• the hydrogel or a mixture of hydrogels and cells and the like used in bioprinting is also referred to as a 'bioink'.
• cells remain specifically located in their original place of placement during the entire culture period, since the cells are unable to maintain or degrade the surrounding alginate gel matrix (Fedorovich, NE et al. Tissue Eng. 13 , 1905-1925 (2007).
• dECM decellularized extracellular matrix
• the ECM of each tissue is unique in terms of composition and topology, and the composition and anatomy are produced through dynamic and interactive interactions between the resident cells and the microenvironment. .
• Recent studies of cells isolated from tissues and organs and ECM highlight the need for tissue specificity to preserve selected cell functions and phenotypes (Sellaro, TL et al. Tissue Eng. Part A 16 1075-1082 (2010); Petersen, TH et al. Science 329, 538-541 (2010); Uygun, BE et al. Nat. Med. 16, 814-821 (2010); Ott, HC et al. Nat. Med. 16, 927-933 (2010); Flynn, LE Biomaterials 31, 4715-4724 (2010)).
• the harvested dECM material is typically processed as a two-dimensional (2D) scaffold from various tissues, including skin, small intestinal submucosa, and in the early stages permeable or seeded cells are supported by supportive vascular networks. It depends on the diffusion of oxygen and nutrients for survival until a supporting vascular network is established.
• Ischemic heart disease has a very high prevalence worldwide and is the number one cause of death for a single disease. Korea is also experiencing a surge in ischemic heart disease due to socioeconomic development and westernized lifestyles. Moreover, in patients with severe end-stage heart failure, there is no cure for heart transplant or mechanical left ventricular assist device challenge. However, there are problems such as lack of donor organs, difficulty in securing long term, high mortality rate and expensive treatment cost.
• stem cell multipotency Treatment using stem cell multipotency is expected to fundamentally treat damaged tissue regeneration.
• adult stem cells are combined with vascular endothelial cells and other stromal cells or mixed with various hydrogels to promote differentiation into cardiomyocytes and prevent cell detachment from lesions, thereby improving the efficiency of heart disease treatment.
• vascular endothelial cells and other stromal cells or mixed with various hydrogels to promote differentiation into cardiomyocytes and prevent cell detachment from lesions, thereby improving the efficiency of heart disease treatment.
• hydrogels to promote differentiation into cardiomyocytes and prevent cell detachment from lesions, thereby improving the efficiency of heart disease treatment.
• There are various researches on heightening In addition, research is being actively conducted to simulate the living environment of stem cells and maximize the interaction between cells by producing cell sheets using stem cells or stem cells and various adult cells.
• tissue-forming structure forming solution comprising a decellularized extracellular matrix and a cross-linking agent
• a system for forming a bioprinting system comprising a cardiac precursor cell, the tissue-forming structure forming solution, a mesenchymal stem cell and Printing and crosslinking the second bioprinting composition including the vascular endothelial growth factor to alternately arrange the first bioprint layer and the second bioprint layer to form a three-dimensional structure; And thermally gelling the cross-linked three-dimensional structure to obtain a cross-gelled three-dimensional structure, the method of manufacturing a three-dimensional structure for myocardial tissue regeneration.
• Another example of the present invention is cross-linking the first bioprint layer and the second bioprint layer prepared by printing the composition for bio-based printing and the low bioprinting composition, and thermally gelling the myocardial tissue. It relates to a three-dimensional structure for.
• the present inventors conducted various studies to develop a method for manufacturing a three-dimensional structure in which the microenvironment of myocardial tissue is implemented. Accordingly, the present inventors have proposed a bioprinting composition comprising decellularized extracellular matrix and cardiac progenitor cells, decellularized extracellular matrix, mesenchymal stem cells, and vascular endothelial growth factor.
• a bioprinting composition comprising decellularized extracellular matrix and cardiac progenitor cells, decellularized extracellular matrix, mesenchymal stem cells, and vascular endothelial growth factor.
• VEGF vascular endothelial growth factor
• the three-dimensional printing method not only equally positions the cardiac progenitor cells in the structure, but also implements a vascular network composed of vascular cells in the structure, thereby maintaining cell viability for a long time, thereby improving cell delivery efficiency into the myocardium. It has been found that it can be significantly improved.
• composition for forming a tissue engineering construct comprising a decellularized extracellular matrix and a crosslinking agent and a cardiac progenitor cell, and a composition for forming a bio-printing system comprising the tissue engineering construct forming solution, a mesenchymal stem cell and a vascular endothelial growth factor Printing and crosslinking with a second composition for bioprinting comprising a first bioprint layer and the second bioprint layer alternately arranged to form a three-dimensional structure Forming;
• It relates to a method of manufacturing a three-dimensional structure for myocardial tissue regeneration comprising a.
• a first bioprinting composition comprising the decellularized extracellular matrix and cardiac progenitor cells as described above;
• a three-dimensional construct prepared by performing a lamination process using alternating CBI bioprinting compositions containing decellularized extracellular matrix, mesenchymal stem cells, and vascular endothelial growth factor is constructed of cardiac progenitor cells. Not only is it evenly located within the structure, but by implementing a vascular network composed of vascular cells in the structure, the microenvironment of the myocardial tissue can be effectively implemented, and the viability of the cells can be maintained for a long time. It can be effected.
• the decellularized extracellular matrix may be derived from cardiac tissue. Specifically, it can be obtained by decellularizing cardiac tissue discharged in vitro, and by decellularizing tissue discharged from mammals such as humans, pigs, cows, rabbits, dogs, goats, sheep, chickens and horses. It may be obtained by decellularizing preferably tissue obtained from pig, more preferably heart tissue obtained from pig, but is not limited thereto.
• Such decellularization may be carried out by known methods such as Ott, H. C. et al. Nat. Med. 14, 213-221 (2008), Yang, Z. et al. Tissue Eng. Part C Methods 16, 865-876 (2010) et al. May be performed using the method disclosed or with minor modifications.
• the decellularization method reported by the inventors namely Falguni Pati, et al., Nat Commun. 5, 3935 (2014) can be performed using the decellularization method.
• the decellularized extracellular matrix can be stored in powder form, usually lyophilized.
• the tax amount of saturated extracellular matrix is not particularly limited, for example, the composition is a total of from 1 to 4 parts by weight 0 / 0,1 to 3 parts by weight 0/0, 2 to 4% by weight relative to the weight, preferably 2 To 3% by weight.
• the decellularized extracellular matrix in the first bioprinting composition is 1 to 4 based on the total weight of the first bioprinting composition. May be present in weight percent;
• the decellularized extracellular matrix in the second bioprinting composition may be present in an amount of 1 to 4% by weight, based on the total weight of the second bioprinting composition. If the content is out of the range is small, the problem that the material is not laminated occurs, if the content is high, there is a problem that the cells enclosed therein die.
• cardiac progenitor cells and vascular endothelial cells are commercially available as cells derived from animals including humans, and can be cultured and expanded by known methods.
• the cardiac progenitor cells are 10 5 to 10 8 cells / ml, 10 6 to 10 8 cells / ml, 10 5 to 5 X 10 7 cells / ml, preferably 10 6 to 5 X 10 in the first bioprinting composition. May exist in the range of 7 cells / ml. If the content is out of the range is low, the efficacy is low, if the content is high, even if the cross-linked bioink-decellularized extracellular matrix surrounding the cells, the decellularized extracellular matrix is not cross-linked three-dimensional The problem of not maintaining the shape occurs.
• the mesenchymal stem cells are 10 5 to 10 8 cells / ml, 10 6 to 10 8 cells / ml, 10 5 to 5 X 10 7 cells / ml, preferably 10 6 to 5 X in the second bioprinting composition. It may be present in the range of 10 7 cells / ml. If the content is out of the range is low, the efficacy is low, if the content is high, even if the cross-linked bioink-decellularized extracellular matrix surrounding the cells, the decellularized extracellular matrix is not cross-linked three-dimensional The problem of not maintaining the shape occurs.
• composition for low U bioprinting and system 2 bioprinting are viscoelastic with a range of pH 6.5 to 7.5 for efficient three-dimensional printing. It is desirable to have the form of a uniform solution.
• the tissue-forming structure forming solution is acid; Protease; And pH adjusters.
• the acid, protease, and pH adjusting agent may be included in the aqueous medium, but is not limited to.
• the acid may be at least one acid selected from the group consisting of acetic acid and hydrochloric acid, but is not limited thereto.
• the acid serves to dissolve the decellularized extracellular matrix, preferably acetic acid, hydrochloric acid, etc.
• aqueous acetic acid solution for example about 0.5 M aqueous acetic acid solution, or 0.01 to 10 M , 0.01 to 9 M, 0.01 to 8 M, 0.01 to 7 M, 0.01 to 6 M, 0.01 to 5 M, 0.01 to 4 M, 0.01 to 3 M, 0.01 to 2 M, 0.01 to 1 M, 0.01 to 0.1 M
• Aqueous hydrochloric acid solution for example in the form of an aqueous hydrochloric acid solution of about 0.01 M.
• the protease performs a digestion function of telopeptide of the decellularized extracellular matrix, and may be at least one selected from the group consisting of pepsin and matrix metalloproteinase, It is not limited to this.
• the amount of the protease is different depending on the content of the decellularized extracellular matrix, for example, 5 to 30 mg, 5 to 25 mg, 10 to 30 mg, preferably 100 mg of the decellularized extracellular matrix. Can be used at a rate of 10 to 25 mg.
• the pH regulator neutralizes the acid used for lysis of the decellularized extracellular matrix, thereby adjusting the pH of the composition to pH 6.5 to pH.5, pH 6.6 to 7.4, pH 6.7 to 7.3, pH 6.8 to 7.2, pH 6.9 to 7.1. , Preferably it is to adjust to the range of pH 7, for example, may be sodium hydroxide, but is not limited thereto.
• the system first bio-printing composition for the composition and the second bio-printing for is independently, for each of the composition total weight, acetic acid, and selected from the group true made with hydrochloric acid at least one acid of 0.03 to 30 parts by weight 0/0 ; 1 to 0.4 wt% of at least one protease selected from the group consisting of pepsin and matrix metalloproteinases; and ⁇ modulators.
• the first bioprinting composition and the second bioprinting composition may each independently include one or more selected from the group consisting of cells, growth factors and enzymes.
• the cell may be at least one selected from the group consisting of endothelial progenitor cells, endothelial cells, and cardiomyocytes.
• the growth factor fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), angiopoietin -l (angiopoietin-l), transforming growth factor -beta (transforming) growth factor beta (TGF- ⁇ ), erythropoietin (EPO), stem cell factor (SCF), epidermal growth factor (EGF) and colony stimulating factor (CSF) It may be one or more selected from the group.
• the enzyme may be at least one selected from the group consisting of matrix metalloproteinases (MMPs) and matrix inhibitors of matrix metalloproteinases (TIMPs).
• MMPs matrix metalloproteinases
• TMPs matrix inhibitors of matrix metalloproteinases
• the first bioprinting composition and the second bioprinting composition are each independently,
• One or more cells selected from the group of cells consisting of endothelial progenitor cells, endothelial cells, and cardiomyocytes; And
• Fibroblast growth factor FGF
• platelet-derived growth factor PDGF
• angiopoietin-l transforming growth factor-beta TGF- ⁇
• erythropoietin EPO
• stem cell factor SCF
• epidermal growth factor EGF
• colony stimulating factor CSF
• first bioprinting composition and the second bioprinting composition further comprising one or more cells and one or more growth factors, each independently,
• the method may further include one or more enzymes selected from the group consisting of matrix metalloproteinases (MMPs) and substrate inhibitor matrix metalloproteinases (TIMPs).
• MMPs matrix metalloproteinases
• TMPs substrate inhibitor matrix metalloproteinases
• the growth factor may be included in the form of endothelial cell growth supplement (ECGS), but is not limited thereto.
• ECGS endothelial cell growth supplement
• the first bioprinting composition and the second bioprinting composition are preferably viscoelastic materials having a lower viscosity as the shear rate increases, for example, at a shear rate 1 when measured at about 15 ° C. It is preferable that the viscosity is in the range of 1 to 30 Pa-S.
• the viscosity can be adjusted by appropriately adjusting the amount of aqueous medium (eg, water, distilled water, PBS, saline, etc.). The crab 1 for bioprinting.
• Printing using the composition and the composition for the second bioprinting alternately uses a known three-dimensional printing method (for example, a printing method using a 'multiaxial tissue organ printing system'), Falguni Pati, et aL, zt Commun . 5, 3935 (2014), etc., and may be performed, for example, using two syringes of a multiaxial tissue organ printing system.
• a known three-dimensional printing method for example, a printing method using a 'multiaxial tissue organ printing system'
• a polycaprolactone (PCL) framework is loaded into a syringe and heated to about 80 ° C to melt the polymer.
• the above composition for three-dimensional printing in the form of pre-gel is loaded in another syringe and the temperature is maintained at about 15 ° C. or lower, preferably about 4 to 10 ° C.
• Pneumatic pressure is applied in the range of 400 to 650 kPa for fabrication of the PCL framework.
• the composition in pre-gel form is sprayed using a plunger-based low-dosage dispensing system.
• printing using the first bioprinting composition and the second bioprinting composition alternately uses a plunger-based low-dose injection system only for the composition in the pre-gel form without using a polycaprolactone framework. It may also be carried out by spraying using a dosage dispensing system.
• the first composition for bioprinting and the second The stack using alternating bioprinting compositions may be stacked such that the first bioprinting composition and the second bioprintant composition are arranged to have a predetermined crossing angle with each other.
• first bioprinting composition and the second bioprinting composition may be printed in the form of fiber, tape, or fabric.
• the first bioprinting composition and the second bioprinting composition are each printed on one layer in the form of a fiber, and the one layer is a fiber and the second bioprinting on which the first bioprinting composition is printed.
• the composition may be a layer made of unidirectional fibers in which the printed fibers are alternated with each other, and another layer may be laminated to have a predetermined crossing angle with the layer. That is, it may have a structure in which one layer of ' 0 degree direction ' and one layer of 90 degree's direction are stacked. An example of such a stacking form is illustrated in FIG. 3.
• the crosslinker may be riboflavin.
• the reason for setting the above range is that the mechanical properties similar to those of the heart tissue are exhibited.
• each lamination process may be performed through the printing and crosslinking under UVA.
• the printing and crosslinking under UVA that is, by performing a layer-by-layer process, a three-dimensional structure shape is formed.
• the crosslinking under UVA may use UVA with a wavelength of 315 to 400 nm, 325 to 390 nm, 335 to 380 nm, 345 to 370 nm, 355 to 365 nm, preferably about 360 nm.
• the crosslinking under UVA is 1 to 10 minutes, 1 to 9 minutes, 1 to 8 minutes, 1 to 7 minutes, 1 to 6 minutes, 1 to 5 minutes, 1 to 4 minutes, 2 to 10 minutes, 2 to 9 minutes, 2 to 8 minutes, 2 to 7 minutes, 2 to 6 minutes, 2 to 5 minutes, 2 to 4 minutes, preferably about 3 minutes.
• a crosslinking-thermal gelation method involving the use of riboflavin.
• crosslinking-thermal gelation method using riboflavin not only can provide high mechanical strength, but also requires the use of toxic crosslinking agents such as glutaraldehyde and also uneven crosslinking inside the three-dimensional structure. The problem of post-print crosslinking can be avoided.
• the three-dimensional structure has a modulus at 1 rad / s of 1 to 100 kPa, 1 to 90 kPa, 1 to 80 kPa, 1 to 70 kPa, 1 to 60 kPa, 1 to 50 kPa, 1 to 40 kPa. , 1 to 30 kPa, 1 to 20 kPa, for example, 10.58 kPa.
• the reason for setting the above range is that it has an effect of simulating the surrounding environment with mechanical strength similar to that of the heart muscle.
• the first bio-printing composition and the second composition for a bio for printing is independently, for each of the total composition weight, from 0.001 to 0.1 increased riboflavin 0/0; Acetic acid, and at least one member from the group true made acid with hydrochloric acid, from 0.03 to 30 parts by weight selected 0 / .; pepsin and the matrix at least one metal from the group consisting of proteinase kinase in the selected protease coming from 1 to 0.4 parts by weight 0/0; And ⁇ modulators.
• composition for crab bioprinting and low 12 bioprinting composition according to the embodiment,
• riboflavin, ⁇ modulator and cardiac progenitor cells for the first bioprinting composition
• riboflavin, ⁇ modulator, mesenchymal stem cells and vascular endothelial growth factor for the second bioprinting composition
• It may be prepared by a manufacturing method comprising a.
• the step of obtaining the solution to which the protease is added may be performed after adding the protease, and the stirring may be performed until the complete lysis of the decellularized extracellular matrix is achieved. Usually 24 to It may be performed for 48 hours, but is not limited thereto.
• the printing and crosslinking may be carried out at 15 ° C or less to prevent gelation, for example, 4 to 15 ° C, preferably at a low temperature of 4 to 10 ° C. .
• the thermal gelation may be performed at more than 15 ° C, for example, 20 ° C or more, 25 ° C or more, preferably, 20 ° C to 50 ° C, 20 ° C to 40 ° C. , 20 ° C to 3TC, 25 ° C to 50 ° C, 25 ° C to 40 ° C, more preferably, may be carried out at a temperature of 25 ° C to 37 ° C.
• Bio-composition and low-12 bioprinting composition prepared by the production method is a pH-adjusted pre-gel form, preferably stored at about 4 ° C. .
• the thermal gelation may be performed at more than 15 ° C, for example, 20 ° C or more, 25 ° C or more, preferably, 20 ° C to 50 ° C, 20 ° C to 40 ° C , 20 ° C to 37 ° C, 25 ° C to 50 ° C, 251 to 40 ° C, more preferably, may be carried out at a temperature of 25 ° C to 37 ° C.
• the reason for setting the above range is because there is an effect of physically crosslinking the decellularized extracellular matrix.
• the thermal gelation can be carried out in a incubator (humid incubator).
• the thermal gelation may be performed for 5 to 60 minutes, 10 to 50 minutes, 15 to 40 minutes, preferably 20 to 30 minutes.
• the reason for setting the range is that it is the range required for the physical crosslinking reaction to occur.
• the thickness of the three-dimensional structure prepared by the method can be adjusted according to the amount of cells to be delivered, 50 to 1000, 50 to 900 urn, 50 to 800 ⁇ m, 50 to 700 ⁇ , 50 to 600 fm, 50 to 500 // m, 100 to 1000; urn, 100 to 900 ⁇ m, 100 to 800 /, 100 to 700, 100 to 600 ⁇ ⁇ ⁇ , 100 to 500 / m, 200 to 1000 ⁇ , 200 to 900, 200 to 800 ⁇ m, 200 to 700 / m, 200 to 600 ⁇ m, 200 to 500; urn, 300 to 1000 ⁇ , 300 to 900 ⁇ m, 300 to 800 ⁇ m, 300 to 700 ⁇ , 300 to 600 ⁇ , 300 to 500 ⁇ ⁇ , 400 to 1000 ⁇ , 400 to 900 ⁇ , 400 to 800 urn, 400 to 700 ⁇ m, 400 to 600 ⁇ m, 400 to 500 m, for example 500 .
• the manufacturing method is to place the cardiac precursor cells evenly in the structure.
• a vascular network consisting of vascular cells in the structure can maintain the viability of the cell for a long time, can significantly improve the efficiency of cell delivery into the myocardium, the cardiac precursor cells are evenly placed in the structure Is shown in FIG. 9.
• Another embodiment of the present invention relates to a three-dimensional structure for myocardial tissue regeneration.
• the second bioprinting composition comprising the leaf stem cells and the vascular endothelial growth factor may include one or more structures alternately stacked by printing and crosslinking.
• the first bioprinting composition, the second bioprinting composition, decellularized extracellular matrix, cardiac progenitor cells, mesenchymal stem cells, vascular endothelial growth factor are the same as described above.
• the structure in which the first bioprinting composition and the second bioprinting composition are alternately laminated by printing and crosslinking may be arranged such that the first bioprinting composition and the second bioprinting composition have a predetermined crossing angle with each other. It may be a stacked structure.
• the first bioprinting composition and the second bioprinting composition may be printed in the form of fiber, tape, or fabric.
• each layer may be printed on one layer of the first bioprinting composition and the second bioprinting composition, respectively, in the form of a heart oil, and the one layer may be formed of fibers and the first bioprinting composition.
• the bioprinting composition may be a layer in which the printed fibers are made in the form of one-way fibers alternated with each other, and another layer may be laminated to have a predetermined crossing angle with the layer. That is, it may be a structure in which one layer in the 0 degree direction and one layer in the 90 degree direction are stacked. The structure in which each layer is stacked once may be about 50 thick.
• the thickness of the three-dimensional structure can be adjusted according to the amount of cells to be delivered, 50 to 1000 ⁇ , 50 to 900 ⁇ , 50 to 800 ⁇ ⁇ ⁇ , 50 to 700 im, 50 to 600 ⁇ m, 50 to 500 m, 100 to 1000 m, 100 to 900 jm, 100 to 800 /, 100 to 700 m, 100 to 600, 100 to 500 ⁇ ⁇ ⁇ , 200 To 1000 ⁇ m, 200 to 900 ⁇ m, 200 to 800 ⁇ , 200 to 700 ⁇ , 200 to 600 /, 200 to 500 ⁇ m, 300 to 1000 ⁇ m, 300 to 900 ⁇ m, 300 to 800, 300 to 700 m, 300 to 600 fm, 300 to 500 m, 400 to 1000, 400 to 900 ⁇ , 400 to 800 ⁇ , 400 to 700 ⁇ , 400 to 600 m, 400 to 500, for example, thickness of 500 It can have
• the present invention relates to bioprinting composition (s) comprising specific cells, namely cardiac progenitor cells and / or mesenchymal stem cells and vascular endothelial growth factor (VEGF).
• bioprinting composition comprising specific cells, namely cardiac progenitor cells and / or mesenchymal stem cells and vascular endothelial growth factor (VEGF).
• the manufacturing method of the present invention can not only position the cardiac progenitor cells evenly in the structure, but also maintain the viability of the cells for a long time by implementing a vascular network composed of vascular cells in the structure, thereby improving cell delivery efficiency into the myocardium. It can be significantly improved. Therefore, the manufacturing method of the present invention can be usefully applied to the production of a three-dimensional structure for the treatment of various heart diseases, including ischemic heart disease. [Brief Description of Drawings]
• hdECM decellularized extracellular matrix
• Figure 2 is an optical micrograph and tissue staining picture of decellularized extracellular matrix (hdECM) obtained from cardiac tissue according to an embodiment of the present invention.
• 3 is a schematic diagram of a three-dimensional structure manufactured according to an embodiment of the present invention.
• Figure 4 is a photograph showing the shape of the three-dimensional structure manufactured using the PCL framework according to an embodiment of the present invention.
• 5 is an echocardiography (echocardiography) m-mode 'photograph taken after implantation of a three-dimensional structure prepared in accordance with one embodiment of the present invention to the rat myocardial infarction, and 8 weeks after the implantation.
• echocardiography echocardiography
• FIG. 6 is a rat three-dimensional structure manufactured according to an embodiment of the present invention After transplantation to the myocardial infarction model, the cardiac function measured before the structure implantation, 4 weeks after transplantation and 8 weeks after transplantation is shown numerically.
• FIG. 8 is a photograph showing the degree of regeneration of the functional myocardium of the myocardial infarction and immunovascular regeneration through immunofluorescence staining according to an embodiment of the present invention.
• FIG. 9 is in the three-dimensional structure for myocardial tissue regeneration prepared in accordance with an embodiment of the present invention . It is a picture showing the heart cells are evenly placed.
• Decellularized extracellular matrix was prepared using Falguni Pati, et al., Nat Commun. 5, 3935 (2014), according to the method disclosed (hereinafter 'hdECM').
• the prepared hdECM was finally freeze-dried and stored frozen until use. 2 shows optical micrographs and tissue staining photographs of the prepared hdECM.
• Pre-Gel (pre- g el) for tissue engineering construct prepared solution was poured in the state of liquid nitrogen produced in the lyophilized hdECM obtained was ground in a mortar and pestle the resulting eu hdECM powder (330 mg) 0.5M
• pepsin (33 mg) (P7125, Sigma-Aldrich) was added and stirred at room temperature for 48 hours.
• riboflavin (2 mg) was added and the pH was adjusted to about pH 7 by dropwise addition of a 10 M NaOH solution angled at 10 ° C or lower.
• the obtained pre-gel The solution for preparing a tissue engineering construct in a state was stored at about 4 ° C.
• the first bioprinting composition was prepared by adding 5 ⁇ 10 6 cells / ml of cardiac precursor cells to the solution for preparing a tissue engineering construct in the pre-gel state.
• the cardiac progenitor cells are obtained from human myocardial tissue-derived cardiac progenitor cells, the Pusan National University School of Medicine, and the Physiology Department.
• Preparation Example 2 Preparation of Second Biolining Composition
• hdECM Decellularized extracellular matrix was prepared using Falguni Pati, et al., Nat Commun. 5, 3935 (2014), prepared according to the method described below (hdECM '). The prepared hdECM was finally freeze-dried and stored frozen until use. 2 shows optical micrographs and tissue staining photographs of the prepared hdECM.
• tissue engineering construct in pre-gel state Preparation of solution The liquid nitrogen was poured into the lyophilized hdECM obtained above, and pulverized with mortar and mortar. After adding hdECM powder (330 mg) to 0.5M aqueous acetic acid solution (10 ml), pepsin (33 mg) (P7125, Sigma-Aldrich) was added and stirred at room temperature for 48 hours. Riboflavin (2 mg) was added while maintaining the temperature of the obtained solution at 10 ° C. or lower, and the pH was adjusted to about pH 7 by dropwise addition of a 10M NaOH solution cooled to KTC or lower. The tissue solution for tissue engineering in the pre-gel state obtained above was stored at about 4 ° C.
• the mesenchymal stem cells 5 X 10 6 cells / ml and vascular endothelial growth factor (product number: 293-VE-lO, R & D systems) in the pre-gel tissue solution for tissue engineering (100 ng / ml) were each added to prepare a composition for second bioprinting .
• the PCL framework polycaprolactone (PCL) framework
• a syringe first syringe
• a multi-axis tissue organ printing system Jin-Hyung Shim et al., J. Micromech.Microeng. 22 085014 (2012)
• the polymer was melted by heating to about 80 ° C.
• the first bioprinting composition of the pre-gel state obtained in Preparation Example 1 was loaded in another syringe (second syringe), and the temperature was maintained at about 10 ° C or less.
• the obtained three-dimensional structure shape was placed in a incubator (humid incubator) of about 37 ° C and maintained for 30 minutes to thermally gel to prepare a three-dimensional structure (referred to as 'CPC printed').
• the resulting three-dimensional structure has a thickness of about 300 to 400 um.
• the three-dimensional structure was manufactured using the composition for system 1 bioprinting and the composition for second bioprinting obtained in Production Examples 1 and 2.
• the PCL framework polycaprolactone (PCL) framework
• a syringe first syringe
• a multi-axis tissue organ printing system Jin-Hyung Shim et al., J. Micromech.Microeng. 22 085014 (2012)
• the polymer was melted by heating to about 8 C.
• the first bioprinting composition in the form of pre-gel obtained in Preparation Example 1 and the bioprinting composition obtained in Preparation Example 2 were prepared in different syringes (2, 3, respectively). Syringe) and the temperature was kept below about 10 ° C.
• the contents of the second syringe were sprayed onto the PCL framework, and crosslinked by irradiation of UVA at about 360 nm for 3 minutes. Thereafter, the content injection of the second and third syringes and the layer-by-layer process through crosslinking were performed to form a three-dimensional structure.
• the obtained three-dimensional structure was placed in an incubator of about 37 ° C. and maintained for 30 minutes to thermally gel to prepare a three-dimensional structure for tissue engineering (called 'CPC / MSC printed'). It has a thickness of about 300 to 400 urn, the shape of which is as shown in FIG. Test Example 1 Measurement of Complex Modulus with or Without Riboflavin
• the cross-linked solution of the pre-gel form obtained in Preparation Example 1 was irradiated for about 360 nm UVA for 3 minutes, and then placed in a humid incubator at about 37 ° C. for 30 minutes to induce thermal gelation. Hydrogels were formed (bridged hydrogel A).
• the preparation solution for tissue engineering constructs in a pre-gel state prepared in the same manner as in Preparation Example 1 was placed in a humid incubator at about 37 ° C. and maintained for 30 minutes to thermally gel. was induced to 'form a hydrogel (hydrogel B).
• the crosslinked hydrogel A according to the present invention has a modulus of 10.58 kPa at 1 rad / s, and is crosslinked by comparison with hydrogel B, which is only gelled without crosslinking. It can be seen that the strength improvement is about 30 times or more.
• Test Example 2 Myocardial infarction was induced by dissecting the chest of 4-5 week-old rats (weight: 300 ⁇ 30 g) and performing permanent ligation of the Left Anterior Descending artery (LAD).
• LAD Left Anterior Descending artery
• LVEF left ventricle ejection fraction
• FS fractional shortening
• the left ventricular inner diameter was significantly reduced compared to the experimental group (control) treated nothing after 4 and 8 weeks of implantation of the three-dimensional structure according to the present invention of Example 3.
• the thickness of the inner wall of the left ventricle 8 weeks after the implantation of the three-dimensional structure prepared according to the present invention was kept thicker than the control and there was less scar tissue formation in the myocardial infarction area.
• the three-dimensional structure (CPC printed) formed by the composition for the first bioprinting alone as in Comparative Example 1 three-dimensional formed with the composition for the first bioprinting and the composition for the second bioprinting as in Example 3
• the scar tissue formation was more effectively suppressed when the construct (CPC / MSC printed) was implanted and the left ventricular inner wall was maintained without thinning.
• a beta representing the contractility of myocardial tissue in the myocardial infarction region implanted with the three-dimensional structure prepared according to the present invention The expression levels of myocin heavy chain (pMHC) and differentiated cluster 31 (CD31) representing angiogenesis were markedly increased, especially 3 formed with low U bioprinting composition and system 2 bioprinting composition. The effect was further maximized when implanting the dimensional structure (CPC / MSC printed) (Example 3).
• the three-dimensional structure produced by the manufacturing method of the present invention can maintain the survival of the cells for a long time by implementing a vascular network (that is, by effectively implementing the microenvironment of the myocardial tissue) into the myocardium It was confirmed that the cell delivery efficiency can be significantly improved.

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